Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information
No repeated dose toxicity study in available on ethyldiisopropylamine (EDIPA). 
However, in a OECD 421 GLP toxicity study was performed with a structural analogue substance,
Dimethylamine.

In an OECD 421 oral screening study for reproductive and developmental effects,
performed according to GLP principles (Charles River, 2021), the parental NOAEL for
Dimethylethylamine was derived to be 80 mg/kg bw/day,
based on adverse stomach findings resulting in mortality
at 150 and 225 mg/kg bw/day. As high dose group (225 mg/kg bw/day)
was prematurely terminated before mating due to the occurrence
of high mortality and clinical signs of toxicity,
both the reproduction and the developmental NOAEL were established
at 150 mg/kg bw/day based on the absence of adverse effects up to and
including 150 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Jul 2020 - 18 Feb 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The objective of this GLP OECD guideline study was to select dose levels for the extended one generation reproductive toxicity study with Dimethylethylamine in rats (Charles River Study No. 20240952).
The potential toxic effects of Dimethylethylamine when given orally by gavage for a minimum of 28 days to Wistar Han rats were determined, and the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development was evaluated. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
version 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
version 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males:11-12 weeks old, females: 13-14 weeks old
- Weight at study initiation: Males: 292 - 352 g; females: 198 - 242 g
- Fasting period before study: No.
- Housing: Animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type) during pregnancy and lactation phase. Animals were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum.
- Water: municipal tap water, ad libitum.
- Acclimation period: 7 days

The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed. It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 47 - 72
- Air changes (per hr): Ten or greater with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained

IN-LIFE DATES: From: 05 Aug 2020 To: 07 Oct 2020
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(Elix)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing formulations (w/w) were prepared at appropriate concentrations to meet dose level requirements using the following method:
water (Elix) was chilled in an ice bath or in the refrigerator before formulating. A stir bar was added to an empty container suitable to hold the final volume with the minimum amount of head space. The scale was tared with the container (plus the seal). The required volume of chilled water (Elix) was transferred to the container. The filled container (plus the seal) was weighed. The test substance was kept cold in a refrigerator or ice bath. A syringe or pipette was used to slowly add the required volume of cold test substance into the chilled water (Elix) under stirring. Part of the required amount of test item (about 70-80%) was added to the vehicle under stirring while the container was in a bath filled with ice water. The container was closed, taken out of the ice water, dried off and placed on the scale to add the remaining amount of test item up to the correct weight. The container was sealed and weighed after this addition. The formulation was stirred magnetically to mix.

The dosing formulations were prepared at least weekly, filled out in daily portions with the use of a syringe or pipette and stored in the refrigerator. The dosing formulations were removed from the refrigerator, stirred at room temperature for at least 30 minutes, and kept at room temperature until dosing. Test substance formulations were dosed within 4 hours after removal from the refrigerator. On the day of dosing, the regular seal was replaced by a seal with a septum / small hole. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Concentration in vehicle: 16, 30, 45 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Details on mating procedure:
- M/F ratio per cage: 1:1 mating
- Length of cohabitation: A maximum of 14 days was allowed for mating
- Proof of pregnancy: appearance of intravaginal copulatory plug or by evidence of sperm in vaginal smear, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy and homogeneity were determined for formulations prepared for use in Week 1, Week 4 and Week 7.

Duplicate samples (approximately 500 mg) taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 50 mL (Group 1 and Group 2) or 100 mL (Group 3 and Group 4). For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations.
Analyses were performed using a validated analytical procedure (Charles River Study No. 20240947).

Concentration results were considered acceptable if mean sample concentration results were within or equal to 85-115% of target concentration. Homogeneity results were considered acceptable if the coefficient of variation was = 10%.

Stability analyses also performed previously in conjunction with the method development and validation study demonstrated that the test item was stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Charles River Study No. 20240947.
Duration of treatment / exposure:
Males of groups 1-3 were treated for 29 days, up to and including the day before scheduled necropsy. Females of groups 1-3 that delivered were treated for 50-63 days, females of groups 1-3 which failed to deliver were treated for 41 days.

Due to severe toxicity observed in animals treated at 225 mg/kg bw/day, resulting in the preterm sacrifice of 3 females on study day 4 and 3 males and 2 females on study day 6, the remaining Group 4 animals were sacrificed for ethical reasons on study day 6 (last dose on day 5).
Frequency of treatment:
once daily, 7 days a week
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Remarks:
group 2 - low dose
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
group 3 - mid dose
Dose / conc.:
225 mg/kg bw/day (actual dose received)
Remarks:
group 4 - high dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral route of administration was selected because this is a possible route of human exposure. The dose levels were selected based on previously performed animal studies with oral administration of Dimethylethylamine in Sprague Dawley rats (i.e. acute oral toxicity study, 14-day preliminary toxicity study, and a OECD 414 study).
During the acute oral toxicity study, an LD50 of 594 mg/kg bw (540-650 mg/kg bw) was determined with 95% confidence intervals. Based on this information, 5 male and female rats received 0, 25, 75 or 225 mg/kg bw/day during the 14-day preliminary toxicity study. No test item-related mortality was observed up to 225 mg/kg bw/day and clinical signs were limited to salivation and chin rubbing in high dose animals. Additionally, the males at 225 mg/kg bw/day had a lower body weight gain (30%) over Days 1-15 of treatment and food consumption of these males was lower during the first week of treatment. The hematology investigation revealed an increased reticulocyte count and red cell distribution width in males and females at 225 mg/kg bw/day, however these findings did not affect the condition or function of the animals. There were no findings attributable to the test item at macroscopic examination, but mean adjusted adrenal weights were 22% higher among males receiving 225 mg/kg bw/day when compared with control. No test item-related effects were observed in the animals treated at 25 or 75 mg/kg bw/day. It was therefore concluded that there were no adverse findings that would preclude the use of at least 225 mg/kg bw/day as the highest dose level for future repeat dose studies.
During the dose range finding study of the OECD 414 study, two rats died on day 3 of treatment when exposed to 360 mg/kg bw/day. The high dose was consecutively reduced to 270 mg/kg bw/day, but all remaining animals died one day later. Histopathology confirmed ulceration in the stomach. During the main OECD 414 study, effects on body weight and food consumption were observed at 180 mg/kg bw/day. Considering all information above, dose levels of 0, 80, 150 and 225 mg/kg bw/day were selected in agreement with the Sponsor.

- Fasting period before blood sampling for clinical biochemistry: Adult males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Females were not fasted overnight.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During treatment, animals were observed at least once daily, up to the day prior
to necropsy. These clinical observations were conducted after dosing at no specific time point.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment
(prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17,
and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
In order to monitor the health status, a few animals were weighed on a few extra occasions.
A terminal weight was recorded on the day of scheduled necropsy.

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined: Yes
Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Thyroid hormone analysis:
Blood of F0-animals (except for high dose group animals and animals which were sacrificed in extremis or found dead) was collected on the day of scheduled necropsy. Samples were collected from the retro-orbital sinus under anesthesia using isoflurane.
Blood samples were collected into tubes without anticoagulant, were processed for serum, and serum was analyzed for Thyroxine (T4) and Thyroid Stimulating Hormone (TSH). Measurement of total T4 and TSH was conducted for F0-males. F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
Assessment of T4 and TSH in F0 females was considered not relevant because there were no test item-related changes in T4 in F0 males, or in the weight and morphology of the thyroid in both sexes.

OTHER:
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.
Sperm parameters (parental animals):
Parameters examined in all adult males: testis weight, epididymis weight.
For the testes of all males in the control and mid dose group, and all males that failed to sire or died before mating (except for males in the high dose group), a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention was paid to the external reproductive genitals which was examined for signs of altered development; gross evaluation of external genitalia.
Live pups were weighed individually on PND 1, 4, 7 and 13. Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight. All male pups in each litter were examined for the number of areola/nipples on PND 13.

Thyroid hormone analysis:
Blood of F1-animals was collected on PND 4 and PND 14-16. On PND 4 at culling, blood was collected from two surplus pups per litter by decapitation and samples were pooled to one sample per litter. On PND 14-16 separate blood samples were collected from two pups per litter. Blood was drawn by aorta puncture under anesthesia. Blood samples were collected into tubes without anticoagulant, were processed for serum, and serum was analyzed for total Thyroxine (T4) and Thyroid Stimulating Hormone (TSH). Measurement of total T4 and TSH was conducted for PND 14-16 pups. Assessment of T4 for PND 4 pups was considered not relevant because no test item-related changes in T4 were noted in pups at PND 14-16.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead, if possible: Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
SACRIFICE
- Parental animals: All animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted. Males (which sired and failed to sire) were euthanized after the completion of the mating period. Females which failed to deliver (with evidence of mating) were euthanized between 25-26 days post-coitum (after confirmation of mating). Females which delivered were euthanized between 14-16 days post partum.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera and the appearance of the tissues and organs was observed macroscopically. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea.

ORGAN WEIGHTS
At necropsy, for all scheduled euthanasia animals, terminal body weight was measured and the following organs were weighed: epididymis, coagulation gland with seminal vesicle gland, thyroid with parathyroid, prostate, testes, adrenal gland, liver and ovaries. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.

-Tissue collection and preservation:
Representative samples of the following tissues were collected from all animals and preserved in 10% neutral buffered formalin: cervix, seminal vesicles including coagulation gland,
mammary gland, thyroid including parathyroid gland, pituitary gland, prostate gland, ovaries, uterus, vagina, adrenal gland, liver, and stomach. Testes and epididymis were preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours. Gross lesions or masses were also collected from all animals if found.

HISTOPATHOLOGY
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
> gross lesions or masses and stomach from all animals, if any
> epididymis, thyroid gland, ovaries, testes, seminal vesicle including coagulation gland, prostate of all animals of the control and mid dose groups and all animals that die spontaneously or are sacrificed in extremis (except for high dose group animals)
> cervix, epididymis, seminal vesicles including coagulation gland, prostate gland, ovaries, uterus,
vagina, testes of all males that failed to sire and females that failed to deliver pups and females with total litter loss.
For the testes of all males in the control and mid dose group, and all males that failed to sire or died before mating (except for high dose group animals) a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Postmortem examinations (offspring):
SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. On PND 4, the surplus pups were euthanized by decapitation. All remaining pups (PND 14-16) except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital. The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

GROSS NECROPSY
Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for
the presence of milk, if possible. If possible, defects or cause of death were evaluated.
For all remaining pups euthanized on PND 14-16, sex was determined both externally and internally.
Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
The thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. If possible, the pups selected for blood sampling were the same pups as selected for thyroid preservation.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE) were reported when possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Datasets with at least 3 groups (control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
For clinical pathology data Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test. The data corresponding to a response variable of interest and to a related covariate was submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons were conducted using Dunnett's test.
Reproductive indices:
Mating index (%): (Number of females mated/Number of females paired) x 100

Precoital time: (Number of days between initiation of cohabitation and confirmation of mating

Fertility index (%): (Number of pregnant females/Number of females mated) x 100

Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites) x 100

Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100

Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/
Number of live pups at First Litter Check) x 100

Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/
Number of live pups at First Litter Check) x 100

Viability index (%): (Number of live offspring on Day 4 before culling/Number live offspring on Day 1
after littering) x 100

Lactation index (%): (Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment at 150 and 225 mg/kg bw/day was associated with clinical signs of toxicity (e.g. calm or lethargic behavior, signs of breathing difficulties, hunched posture and/or piloerection).

At 150 mg/kg bw/day, signs of breathing difficulties (i.e. labored/shallow breathing and/or rales) and piloerection (females only) were also observed in several animals that survived up to scheduled necropsy. Rales and piloerection occurred on incidental occasions with a maximum of seven consecutive days during the treatment period, whereas labored and shallow breathing were seen in two animals on one day only.

At 80 mg/kg bw/day, no clinical signs were observed in males. Piloerection and rales were noted
in four and one female, respectively. Both clinical signs were transient and occurred on 1-5 consecutive days during the treatment period. The salivation seen after dosing among treated animals throughout the dosing period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.

Any other clinical signs noted during the treatment period (e.g. fissures, wounds and scabs) occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
Test item-related mortality occurred in all treatment groups.

At 225 mg/kg bw/day, three males and five females were sacrificed in extremis on Study Days 4 or 6 based on severe weight loss (between 10-20%) when compared with Study Day 1. The sacrificed females (except for one female with no findings), showed next to salivation multiple clinical signs of toxicity (i.e. calm and lethargic behavior, difficulties with breathing (rales, labored respiration and/or gasping), hypothermia, pale appearance and/or piloerection) while observations for the males were
limited to slight salivation.
Based on this mortality and as body weight loss was observed for the majority of the remaining animals at 225 mg/kg bw/day as well (though less severe than the animals sacrificed in extremis), remaining animals were sacrificed on Study Day 6 for welfare reasons. The stomach alterations recorded at necropsy, consisting of foci in the glandular mucosa and/or forestomach, thickened limiting ridge and/or forestomach, yellowish discoloration of the glandular mucosa and/or irregular surface of the forestomach, were regarded to be the main cause of the poor condition of these animals. These animals were sacrificed before mating and no histopathology was performed on these animals.

At 150 mg/kg bw/day four males and two females were euthanized between Day 6 and Day 33 of the study. Several of these animals were sacrificed based on moderate to severe weight loss (between 9-14%) when compared with Study Day 1, but two males were sacrificed due to severe signs of breathing difficulties (i.e. rales, gasping and/or labored/shallow breathing). In addition, one male and one female showed multiple clinical signs of toxicity prior to sacrifice (i.e. lethargy, piloerection, hunched posture and/or a pale appearance). A limited list of organs (reproductive organs, thyroid gland and gross findings) were examined histopathologically. Based on the macroscopic and microscopic findings, the main cause of moribundity was regarded glandular erosions and/or inflammatory lesions in the stomach. Stomach alterations included minimal-slight inflammation of the glandular mucosa (correlating to an irregular surface of the glandular mucosa in one male), slight to marked erosion of the glandular mucosa, minimal-moderate hemorrhage (correlating to reddish/dark-red foci on the glandular mucosa in four males and two females) and/or slight edema of the glandular mucosa. In addition, in the forestomach of a few animals at 150 mg/kg bw/day slight-moderate inflammation and/or slight hyperplasia of the squamous epithelium was recorded.

One female at 150 mg/kg bw/day was found dead on Day 41 of the study. This animal had lost 6% body weight between Study Days 40 and 41 and presented with signs of breathing problems (i.e. gasping and rales) and piloerection on Day 41. This female also showed slightly uncoordinated movements on Days 6-7 of treatment and a slightly tilted head on Days 6-8 and 35-41 of treatment. No cause of death could be determined from the tissue sections examined.

At 80 mg/kg bw/day one male was euthanized on Study Day 6, based on severe weight loss (16%). No clinical signs were observed on the days prior to sacrifice. A limited list of organs (reproductive organs, thyroid gland and gross findings) were examined histopathologically. Based on the macroscopic and microscopic findings, the main cause of moribundity was regarded glandular erosions in the stomach. Stomach alterations included minimal erosion of the glandular mucosa and moderate hemorrhage (correlating to reddish/dark-red foci on the glandular mucosa).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment at 225 mg/kg bw/day was associated with severe body weight loss (between 10-20%).

Treatment at 150 mg/kg bw/day was associated with severe body weight loss for two males
and one female that were sacrificed prior to scheduled necropsy. Several remaining males at 150 mg/kg bw/day showed a slight weight loss or a lower body weight gain when compared with controls, mainly during the first week of treatment. However, individual body weight gains of males surviving up to scheduled necropsy were similar to concurrent control during the last two weeks of treatment (except for a single male). Weight loss was observed in some of the remaining females as well, mainly during the first week of treatment. Individual weight gain values were similar to concurrent control values towards the start of the mating period and during the post-coitum period and beginning of the lactation period. However, body weight gain on PND 13 of females surviving up to scheduled necropsy was noticeably lower when compared with concurrent control values (mean weight gain of 9% vs. 18%, respectively).

Weight gain of females at 80 mg/kg bw/day was similar to concurrent control during the premating, post-coitum and beginning of lactation period. However, body weight gain on PND 13 of 3/7 lactating females was noticeably lower when compared with other females of the same group and concurrent control. Individual values of these three females were similar to values determined for females treated at 150 mg/kg bw/day and were therefore considered test item-related. No test item-related changes in body weights and body weight gain were observed in males at 80 mg/kg bw/day that survived up to scheduled necropsy.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was lower for males and females treated at 150 mg/kg bw/day during the first week of treatment. Mean relative food consumption was 12 and 13% lower when compared with concurrent control for males and females, respectively (not statistically significant). This could most likely be attributed to the animals that were sacrificed in extremis. In subsequent intervals, food consumption was similar to the control level.

Food consumption before or after correction for body weight was similar to the control level at 80 mg/kg bw/day.
Endocrine findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were noted in serum levels of T4 and TSH in F0-males at any dose level.
In F0-males at 150 mg/kg bw/day, mean TSH concentration was increased when compared with
concurrent control (1.69x). No statistical significance was achieved. The higher mean value was attributed to the relatively high value of a single animal (1.490 mU/L). After excluding this animal, a mean value of 0.0696 mU/L (0.38x of control) was obtained. As this change was observed in absence of a corroborative change in T4, it was considered not biologically relevant.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic stomach findings consisted of an increased incidence and severity of alterations in the glandular mucosa to include mixed cell inflammation, erosions, hemorrhage and/or edema in males at 80 and 150 mg/kg bw/day and females at 150 mg/kg bw/day. In some cases the hemorrhage was associated with the glandular erosions.
In the forestomach, findings were seen in a few animals at 150 mg/kg bw/day and included mixed
cell inflammation, ulceration and/or hyperplasia of the forestomach. In the duodenum, present in the stomach section of a single male at 150 mg/kg bw/day, a marked ulcer of the duodenum was recorded.
There were no other test item-related macroscopic or microscopic changes. The remainder of the recorded findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Reproductive Performance:
All cohabitated females showed evidence of mating, however, one control female and three females at 80 mg/kg bw/day were not pregnant. The reproductive organs of males that failed to sire (due to early sacrifice or if cohabitated female was not pregnant) and females that failed to deliver healthy pups were examined histopathologically.

There was 1/10 couples of the control group and 2/10 couples of the 80 mg/kg bw/day group without implantation sites. Additionally, a single female at 80 mg/kg bw/day had no implantation sites. No abnormalities were seen in the reproductive organs of these animals, which could account for their lack of offspring. As all females at 150 mg/kg bw/day were pregnant with implantation sites, the incidence of non-pregnant females at 80 mg/kg bw/day was considered not test item-related in absence of a dose-related response.

Several animals we euthanized prior to mating, one male at 80 mg/kg bw/day, and two males and one female at 150 mg/kg bw/day. There were no findings recorded for the reproductive organs of these animals suggestive of infertility. One female at 150 mg/kg bw/day) was euthanized before delivery and showed features of normal pregnancy.

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item up to 150 mg/kg bw/day.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for one female at 80 mg/kg bw/day. Given the incidental nature and absence of a dose-related response, this finding did not indicate a direct relation with the test item.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was not affected by treatment with the test item up to 150 mg/kg bw/day. All cohabitated females showed evidence of mating, resulting in a mating index of 100% for the control, 80 and 150 mg/kg bw/day groups.

Precoital time was considered not to be affected by treatment with the test item up to 150 mg/kg bw/day. All females showed evidence of mating within 4 days, except for one female at 150 mg/kg/day that showed evidence of mating on Day 12 of the mating period. As this also occurs in control animals from time to time and it concerns one animal only, it was considered incidental and unrelated to treatment with the test item.

Fertility index was considered not to be affected by treatment with the test item up to 150 mg/kg bw/day. The fertility indices were 90, 70 and 100% for the control, 80 and 150 mg/kg bw/day groups, respectively. A total of three females at 80 mg/kg bw/day were not pregnant. Since these cases of nonpregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity in this study, this was considered not to be related to treatment with the test item.

Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were not affected by treatment with the test item up to 150 mg/kg bw/day. All surviving females that were pregnant had live offspring.

No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Number of implantation sites was considered not to be affected by treatment with the test item up to 150 mg/kg bw/day.
The mean number of implantation sites was significantly lower at 80 and 150 mg/kg bw/day when compared with concurrent control. However, when compared with historical control data (mean: 12.3, P5-P95:6.0-16.0 (n=1511), it was found that the concurrent control mean was actually relatively high. One female of the 80 and 150 mg/kg/day groups each had two and six implantation sites only, respectively, which is also sometimes seen in control females, and the number of implantation sites of other females were within normal limits. The number of implantation sites was therefore considered unaffected by the test item.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: as the 225 mg/kg bw/day group was terminated on Study Day 6, no reproduction data is available for this group.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental Systemic toxicity
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Based on adverse stomach findings resulting in mortality at 150 and 225 mg/kg bw/day.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment with
the test item up to 150 mg/kg bw/day.
At 150 mg/kg bw/day, one pup had a paralyzed left hindleg from PND 7 onwards and one other pup from another litter had a swollen left frontleg between PND 2-11. At the incidence observed, no relationship with the test item is indicated.
Additionally, one control pup was sacrificed in extremis on PND 4 as it appeared dehydrated and skinny, with a limited amount of milk in its stomach.

The nature and incidence of other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment with the test item up to 150 mg/kg bw/day.

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment with the test item up to 150 mg/kg bw/day. The live birth index was 99, 100 and 99% for the control, 80 and 150 mg/kg bw/day groups, respectively.

One pup of the control group and one pup at 150 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

The number of live offspring on day 4 before culling compared to the number of offspring on day 1 was not affected by treatment with the test item up to 150 mg/kg bw/day.
Viability index (number of live offspring on PND4 before culling as percentage of number of live offspring on PND1) was not affected by treatment with the test item up to 150 mg/kg bw/day. Viability indices were 99% for the control and 80 mg/kg bw/day groups and 88% for the 150 mg/kg bw/day group. This apparent lower viability index and the statistically significantly higher postnatal loss noted at 150 mg/kg bw/day could mainly be attributed to the death of a single dam and consequently sacrifice of the remaining eight live pups in this litter on day 2 of lactation. When excluding these sacrificed pups, a viability index of 98% is obtained.
Additionally, one pup of the control group, one pup at 80 mg/kg bw/day and two pups at 150 mg/kg bw/day were sacrificed in extremis, found dead or missing on PND 2 or 4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age.

The number of live offspring on Day 13 after littering compared to the number of live offspring on day 4 (after culling) was unaffected by treatment with the test item up to 150 mg/kg bw/day. No pups were found dead/missing between lactation days 5 and 13, resulting in a lactation index of 100% for all groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups determined between Day 1-13 of lactation were considered not to be affected by treatment with the test item up to 150 mg/kg bw/day.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 and TSH levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item up to 150 mg/kg bw/day.
Male pups of two litters at 80 mg/kg bw/day had relatively high TSH levels when compared with concurrent and historical controls. As this change was observed in absence of a dose-response and a corroborative change in T4, it was considered not related to the test item.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item up to 150 mg/kg bw/day.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment with the test item up to 150 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment with the test item up to 150 mg/kg bw/day.
At 150 mg/kg/day, one pup had a paralyzed left hindleg. At the incidence observed, no relationship with the test item is indicated. The nature and incidence of other macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with the test item.
Other effects:
no effects observed
Description (incidence and severity):
Litter size was considered not affected by treatment with the test item up to 150 mg/kg bw/day. A statistically significantly lower mean number of living pups/litter was recorded at 80 and 150 mg/kg bw/day (10.0 and 10.1 vs. 12.9 in the control group). The higher mean of the concurrent control group was however attributed to the relative high number of implantation sites in this group. As a dose-related response was absent and the mean remained within the range considered normal for rats of this strain and age, the lower litter size at 80 and 150 mg/kg bw/day was considered unrelated to treatment with the test item.

Additionally, sex ratio was considered not to be affected by treatment with the test item up to 150 mg/kg bw/day.

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item up to 150 mg/kg bw/day. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 91, 93 and 85% for the control, 80 and 150 mg/kg bw/day groups, respectively.
The slightly lower post-implantation survival index of females at 150 mg/kg bw/day was considered unrelated to treatment with the test item, all individual values remained within the range considered normal for rats of this strain and age.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of developmental toxicity effects at dose levels up to and including150 mg/kg bw/ day.
Remarks on result:
other: as the 225 mg/kg bw/day group was terminated on Study Day 6, no developmental data is available for this group.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Dose Formulation Analysis:


Accuracy:
A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation prepared for use in Weeks 4 and 7. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks. The maximum contribution to the Group 2 samples was 0.019%. In the formulations of Group 1 for use in Week 1, no test item was detected.
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%) except for the Group 2 formulations prepared for use in Week 4 (mean accuracy of 76%). No analytical reason was found for this out of specification result. As there was no indication of structural preparation issues based on the Weeks 1 and 7 results, the lower accuracy found for Group 2 during study Week 4 was considered to be of no negative impact on the study outcome.


Homogeneity:
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).


 


Summary Test Item-Related Macroscopic Stomach Findings - Including (Premature Decedents)














































































































 



Males



Females



Dose level (mg/kg/day):



0



80



150



0



80



150



 



 



 



 



 



 



 



STOMACH a



10



9 (1)



6 (4)



10



10



7 (3)



 



 



 



 



 



 



 



    Foci glandular mucosa



-



1 (1)



1 (4)



-



-



1 (2)



 



 



 



 



 



 



 



     Irregular surface glandular mucosa



-



-



(1)



-



-



 



 



 



 



 



 



 



 



     Irregular surface forestomach



-



-



-



-



-



1



 



 



 



 



 



 



 



    Thickened limiting ridge



-



-



(2)



-



-



(1)



a  =  Number of tissues examined from each group: surviving animals, followed by (..) number of premature decedents


 


Summary Test Item-Related Microscopic Stomach Findings - Including (Premature Decedents)





























































































































































































































































































































 



Males



Females



Dose level (mg/kg/day):



0



80



150



0



80



150



 



 



 



 



 



 



 



STOMACH a



10



9 (1)



6 (4)



10



10



7 (3)



    Inflammation glandular mucosa



 



 



 



 



 



 



        Minimal



1



4



3 (2)



1



3



3



       Slight



-



1



2 (1)



-



-



1 (1)



       Moderate



-



-



1



-



-



1



 



 



 



 



 



 



 



     Erosion glandular mucosa



 



 



 



 



 



 



       Minimal



-



1 (1)



1



-



-



-



       Slight



-



-



-



-



-



1 (1)



       Moderate



-



-



-



-



-



1 (1)



       Marked



-



-



(1)



-



-



-



 



 



 



 



 



 



 



     Hemorrhage glandular mucosa



 



 



 



 



 



 



       Minimal



-



1



(3)



-



-



-



       Slight



-



-



1



-



-



(1)



       Moderate



-



(1)



(1)



-



-



1 (1)



 



 



 



 



 



 



 



       Edema glandular mucosa



 



 



 



 



 



 



       Minimal



-



1



1



-



-



-



       Slight



-



-



(1)



-



-



(1)



 



 



 



 



 



 



 



     Inflammation forestomach



 



 



 



 



 



 



       Slight



-



-



-



-



-



(1)



       Moderate



-



-



(1)



-



-



1



 



 



 



 



 



 



 



     Ulcer forestomach



 



 



 



 



 



 



       Moderate



-



-



-



-



-



1



 



 



 



 



 



 



 



     Hyperplasia squamous epithelium


                                     forestomach



 



 



 



 



 



 



       Minimal



-



-



-



-



-



2



       Slight



-



-



(1)



-



-



-



 



 



 



 



 



 



 



a  =  Number of tissues examined from each group: surviving animals, followed by (..) number of premature decedents


 


 

Conclusions:
In an oral screening study for reproductive and developmental effects, performed according to GLP principles, the parental NOAEL for Dimethylethylamine was derived to be 80 mg/kg bw/day, based on adverse stomach findings resulting in mortality at 150 and 225 mg/kg bw/day. As high dose group (225 mg/kg bw/day) was prematurely terminated before mating due to the occurrence of high mortality and clinical signs of toxicity, both the reproduction and the developmental NOAEL were established at 150 mg/kg bw/day based on the absence of adverse effects up to and including 150 mg/kg bw/day.
Executive summary:

The potential toxic effects of Dimethylethylamine when given orally by gavage at 80, 150 and 225 mg/kg/day for a minimum of 28 days to Wistar Han rats were determined, and the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development was evaluated up to at least PND 13.


At 225 mg/kg/day, mortality and clinical signs of toxicity (e.g. calm or lethargic behavior, signs of breathing difficulties, hypothermia, pale appearance and/or piloerection) were observed. In total, eight animals were sacrificed in extremis between Study Day 4 and 6 based on severe body weight loss and/or severe clinical signs. Due to this mortality and as body weight loss was observed for the majority of the remaining animals at this dose level as well (though less severe than the animals sacrificed in extremis), this group was terminated on Study Day 6 for animal welfare reasons. The alterations in the stomach observed at necropsy in all decedents were considered to be test item-related, adverse and the main cause of the poor condition of these animals.


At 150 mg/kg/day, mortality and clinical signs of toxicity (e.g. lethargic behavior, signs of breathing difficulties, hunched posture, pale appearance and/or piloerection) were observed. In total, six animals were sacrificed in extremis between Study Day 6 and 33 based on severe body weight loss and/or severe clinical signs. The alterations in the stomach observed at necropsy in all decedents were considered to be test item-related, adverse and the main cause of the poor condition of these animals. Macroscopic findings were correlated microscopically with glandular erosions and/or inflammatory lesions in the stomach. For one female, found dead on Study Day 41, no clear cause of death could be revealed. For several remaining animals at 150 mg/kg/day, slight body weight loss or lower body weight gain was observed mainly during the first week of treatment. Additionally, a lower body weight gain was observed in females on PND 13 when compared with control. Animals treated at 150 mg/kg/day that survived up to scheduled necropsy showed similar adverse stomach alterations as the preterm sacrifices of the 150 and 225 mg/kg/day groups.


Based on the adverse stomach findings, which were considered the cause of the moribundity observed at 150 and 225 mg/kg/day, both dose levels were considered inappropriate as the high dose for the subsequent OECD 443 study.


At 80 mg/kg/day, one male was sacrificed in extremis on Study Day 6 based on severe weight loss. In the stomach of this decedent, moderate hemorrhage associated with a minimal erosion of the glandular mucosa was observed. In males surviving until scheduled necropsy, findings in the glandular stomach were recorded at low severities. Clinical signs (i.e. rales and piloerection) were observed in females only, were transient and occurred during a short period of time only. In 3/7 lactating females, body weight gain on PND 13 was noticeably lower than other females of the same group and concurrent control. Individual values of these three females were similar to values determined for females treated at 150 mg/kg/day and were therefore considered test item-related. However at the incidence observed and absence of effects on pup development, this change was considered not dose-limiting. As no further toxicity was observed except for the single mortality and minimal stomach findings, a dose level of 80 mg/kg/day was considered feasible as high dose for the subsequent OECD 443 study.


No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical biochemistry (male T4 and TSH thyroid hormone levels) and organ weights) at 80 and 150 mg/kg/day in F0-animals. 


No reproductive or developmental toxicity was observed up to 150 mg/kg/day.


No test item-related changes were noted in any of the reproductive or developmental parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, histopathological examination of reproductive organs, gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 and TSH thyroid hormone levels and macroscopic examination).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No data was generated on ethyldiisopropylamine (EDIPA) for the effects on fertility. However, a combined repeat dose and reproductive/developmental toxicity screening test was performed with a structural analogue substance, Dimethylamine (CAS No. 598-56-1) where no alert on fertility/reproduction were seen:


The potential toxic effects of Dimethylethylamine when given orally by gavage at 80, 150 and 225 mg/kg/day for a minimum of 28 days to Wistar Han rats were determined, and the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development was evaluated up to at least PND 13.


At 225 mg/kg/day, mortality and clinical signs of toxicity (e.g. calm or lethargic behavior, signs of breathing difficulties, hypothermia, pale appearance and/or piloerection) were observed. In total, eight animals were sacrificed in extremis between Study Day 4 and 6 based on severe body weight loss and/or severe clinical signs. Due to this mortality and as body weight loss was observed for the majority of the remaining animals at this dose level as well (though less severe than the animals sacrificed in extremis), this group was terminated on Study Day 6 for animal welfare reasons. The alterations in the stomach observed at necropsy in all decedents were considered to be test item-related, adverse and the main cause of the poor condition of these animals.


At 150 mg/kg/day, mortality and clinical signs of toxicity (e.g. lethargic behavior, signs of breathing difficulties, hunched posture, pale appearance and/or piloerection) were observed. In total, six animals were sacrificed in extremis between Study Day 6 and 33 based on severe body weight loss and/or severe clinical signs. The alterations in the stomach observed at necropsy in all decedents were considered to be test item-related, adverse and the main cause of the poor condition of these animals. Macroscopic findings were correlated microscopically with glandular erosions and/or inflammatory lesions in the stomach. For one female, found dead on Study Day 41, no clear cause of death could be revealed. For several remaining animals at 150 mg/kg/day, slight body weight loss or lower body weight gain was observed mainly during the first week of treatment. Additionally, a lower body weight gain was observed in females on PND 13 when compared with control. Animals treated at 150 mg/kg/day that survived up to scheduled necropsy showed similar adverse stomach alterations as the preterm sacrifices of the 150 and 225 mg/kg/day groups.


Based on the adverse stomach findings, which were considered the cause of the moribundity observed at 150 and 225 mg/kg/day, both dose levels were considered inappropriate as the high dose for the subsequent OECD 443 study.


At 80 mg/kg/day, one male was sacrificed in extremis on Study Day 6 based on severe weight loss. In the stomach of this decedent, moderate hemorrhage associated with a minimal erosion of the glandular mucosa was observed. In males surviving until scheduled necropsy, findings in the glandular stomach were recorded at low severities. Clinical signs (i.e. rales and piloerection) were observed in females only, were transient and occurred during a short period of time only. In 3/7 lactating females, body weight gain on PND 13 was noticeably lower than other females of the same group and concurrent control. Individual values of these three females were similar to values determined for females treated at 150 mg/kg/day and were therefore considered test item-related. However at the incidence observed and absence of effects on pup development, this change was considered not dose-limiting. As no further toxicity was observed except for the single mortality and minimal stomach findings, a dose level of 80 mg/kg/day was considered feasible as high dose for the subsequent OECD 443 study.


No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical biochemistry (male T4 and TSH thyroid hormone levels) and organ weights) at 80 and 150 mg/kg/day in F0-animals. 


No reproductive or developmental toxicity was observed up to 150 mg/kg/day.


No test item-related changes were noted in any of the reproductive or developmental parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, histopathological examination of reproductive organs, gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 and TSH thyroid hormone levels and macroscopic examination).


 


 

Effects on developmental toxicity

Description of key information

A GLP rat developmental study (OECD 414) was performed with EDIPA (Covance, 2021). The maternal No-Observed-Adverse-Effect-Level (NOAEL) was 30 mg/kg bw/day, based on the adverse loss in maternal body weight during the first two days of treatment (Days 6 and 7 of gestation), the adverse low adjusted body weight change and the low food consumption during Days 6-10 of gestation at 80 mg/kg/day. The embryo-fetal No-Observed-Adverse-Effect-Level (NOAEL) was 80 mg/kg bw/day. No developmental effects were noted with EDIPA.


In addition a supportive GLP OECD 414 developmental study was performed with the analogue DMEA (Covance, 2020).The no observed adverse effect level (NOAEL) for maternal toxicity was 60 mg/kg/day based on the effect on maternal body weight and food consumption. For embryo fetal survival and development the NOAEL was considered to be 180 mg/kg/day. No alert on embyrofetal development was noted with DMEA.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date (animal arrival) 02 September 2020
Experimental completion date (fetal pathology) 16 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: • Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Duration of acclimatization Five days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation) 69 to 75 days old.
Weight range of the animals at the start of the study (Day 0 of gestation) 226 to 276 g.


Animal Care and Husbandry
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24¿C and 40-70%.

There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light: 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.

Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.

Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.

Cage distribution The cages constituting each group were blocked by group and mounted in batteries.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Acclimatization up to four animals
During pairing one (stock) male and one female
Gestation one female

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.

Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified pelleted diet.

The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen chew blocks.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.
Route of administration:
oral: gavage
Vehicle:
other: 1% w/v methylcellulose plus 0.1% Tween 80.
Details on exposure:
Method of preparation The required amount of test item was weighed and approximately 50% of the required volume of vehicle was added and it was magnetically stirred until it was uniformly mixed. The pH was adjusted with 37% HCl to pH 7.0-8.0. It was then made up to the required volume with vehicle and magnetically stirred for a minimum of 20 minutes.
A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation Weekly.
Storage of formulation Refrigerated (2 to 8°C) for up to eight days and ambient (15 to 25°C) for 24 hours.
Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulation Analysis
Stability and homogeneity The homogeneity and stability of formulations during storage were confirmed as part of another study, Covance Study Number LB21BK.
Achieved concentration Samples of each of the first, second and last formulation preparations weeks were analyzed for achieved concentration of the test item.
Sample handling Samples taken following formulations in the second and last weeks were added directly into volumetric containers containing water.

Liquid chromatograph: Waters Acquity UPLC
Mass spectrometer: Waters TQC
Ion source: Positive electrospray
Analytical column: Waters Acquity UPLC BEH C18 1.7 µm, (5 cm ¿ 2.1 mm)
Column temperature: 45°C
Mobile phase A: Water:methanol:formic acid (90:10:0.1 v:v:v) + 0.01 M ammonium formate

Gradient (isocratic): Time flow (mL/min) %A
(mins)
0 0.4 100
4.0 0.4 100

Injection volume: 20 µL (full loop)
Strong injector wash : Water/methanol/acetonitrile/isopropanol 25/25/25/25 v/v/v/v
Weak injector wash : Water/methanol 90/10 v/v

Details on mating procedure:
Mating
Male/female ratio 1:1 with identified stock males.

Daily checks for evidence of mating Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.

Day 0 of gestation When positive evidence of mating was detected.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Allocation and Identification
Allocation On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

Method To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.

Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted subcutaneously in the dorsal cervical region.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.
Duration of treatment / exposure:
Day 6 to 19 of mating
Frequency of treatment:
Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.
Duration of test:
Day 0-6: Mating
Day 6-19: Treatment
Day 20: Necropsy
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control - vehicle only
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
80 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Animal Model
The rat (virgin) was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Route of Administration
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Rationale for Dose Level Selection
The dose levels selected for investigation in this main study for effects on embryo-fetal development (0, 10, 30 and 80 mg/kg/day) were selected in conjunction with the Sponsor and were based on the results of a preliminary study for effects on embryo-fetal development study (Covance Study No. 8434891). In that study, pregnant female Crl:CD(SD) rats received Ethyldiisopropylamine at doses of 25, 50 and 100 mg/kg/day. A similarly constituted control group received the vehicle, 1% w/v methylcellulose plus 0.1% Tween 80, at the same volume dose as control groups.

In the preliminary study, clinical signs were generally evident for females that received 100 mg/kg/day comprising decreased activity, rales, piloerection, partially closed eyelids, prominent eyes, uncoordinated gait and whole body pallor. There were no treatment related clinical signs for females that received 25 or 50 mg/kg/day. Furthermore, body weight losses were evident in the 100 mg/kg/day group and group mean body weight gain for this group was low when compared to the controls. For females that received 25 or 50 mg/kg/day, there was a suggestion of slightly reduced body weight gain when compared to the controls. Group mean food intake was reduced for females that received 100 or 50 mg/kg/day when compared to the controls and there was no effect of treatment on food intake for females that received 25 mg/kg/day.

For females that received 100 or 50 mg/kg/day, there were no abnormalities of the dams or fetuses at the macroscopic examination.
Maternal examinations:
Serial Observations
Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:
• Pre-dose observation
• One to two hours after completion of dosing
• As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

Body Weight
The weight of each adult was recorded on Days 0, 3 and 6 to 20 after mating.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

Thyroid Hormone Analysis
Blood samples were collected at the following occasion:
Occasion Animals
At termination All adult females

Parameters Triiodothyronine (T3)
Thyroxine (T4)
Thyroid stimulating hormone (TSH)
Sequence of blood sampling on each occasion To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons.

Conditions No overnight deprivation of food.
Blood sample site Sublingual vein.
Anesthetic Isoflurane.
Anticoagulant None.
Tubes Greiner Minicollect tubes with clotting activator.
Blood volume 1.0 mL.
Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.
Number of aliquots Two per animal. Aliquot 1: 0.2 mL serum for T3/T4
Aliquot 2: residual serum for TSH
Final storage conditions Deep frozen (approximately -60°C to -80¿C) pending analysis.
Fate of samples Aliquot 1 (T3 and T4): dispatched to the Department of Bioanalysis, Covance.
Aliquot 2: dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.
T3 and T4 Performed by the Department of Department of Bioanalysis, Covance.
The method of analysis and results are presented in Attachment 13.3.
TSH Performed by the Department of Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Schedule Animals surviving until the end of the scheduled study period were killed on Day 20 after mating.

Sequence To allow satisfactory inter-group comparison.

Organ Weights
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.

Thyroid and abnormalities All adult females.
Routine staining Sections were stained with hematoxylin and eosin.

Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Scheduled kill All animals from all groups. Thyroid gland only.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
Ovaries and uterine content:
The following were recorded for all animals:
Uterus Gravid uterine weight (including cervix and ovaries).

For each ovary/uterine horn Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).

Fetal examinations:
Fetal Examination and Processing
Examination of all viable fetuses and placentae Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Fetuses examined externally with abnormalities recorded. The sex and ano-genital distance of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter Sexed internally and eviscerated.

Fixation Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS).

Remaining fetuses were fixed whole in Bouin’s fluid.

Processing Bouin’s fixed fetuses were subject to free-hand serial sectioning.

IMS fixed fetuses were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed fetuses Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses Assessed for skeletal development and abnormalities.

Fetal, Litter and Placental Weights
Mean fetal weights were calculated for each litter. Values were presented for male, female and overall fetal weight. Litter weight was calculated as the sum of all fetal weights. Mean placental weight was also calculated for each litter.

Group mean values and SD were calculated using individual litter mean values.

Ano-Genital Distance
Ano-genital distance were presented both as absolute/unadjusted and adjusted for fetal body weight, using the weight recorded at necropsy.

Detailed Fetal Examination
Findings from external, visceral and skeletal examination of fetuses are tabulated on an individual basis for affected litters and fetuses, linking the results of initial external examinations with subsequent visceral and/or skeletal examinations to fetal weight.

Group incidences of observations on fetuses and litters are summarized in terms of major or minor abnormalities or as skeletal variants. The incidence of structural changes are presented as numeric fetal and litter incidences.

Findings observed were classified, according to severity and incidence, as:

Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect.

Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.

Variants: alternative structures or stages of development occurring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.

Observations on repeated structures like ribs, vertebrae and sternebrae are reported as the first and last affected element, in the form ‘5th 13th bilateral ribs’, which should be interpreted as ‘5th to 13th bilateral ribs’.
Statistics:
Please refer to "Any other information on materials and methods"
Indices:
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations) / Number of corpora lutea x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = (Number of implantations - Number of live fetuses) /Number of implantations x 100

All group values and SD were calculated from the individual litter values.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs associated with treatment were evident from Day 6 of gestation, at 30 or 80 mg/kg/day. There were no signs associated with dosing at 10 mg/kg/day.

Partially closed eyelids, with or without piloerection were observed in several animals at 1 to 2 hours after dosing at 30 or 80 mg/kg/day that persisted in some animals at the end of the day and were evident in some animals at each dose before dosing on Days 17 19. The incidence of these signs tended to increase during the treatment period. In addition, underactive behaviour was evident on Day 6 at one to two hours after dosing in one animal (No. 54) at 30 mg/kg/day and one animal (No. 72) at 80 mg/kg/day and on Day 7 in one animal (No. 65) at 80 mg/kg/day. One animal (No. 79) at 80 mg/kg/day had hunched posture on Day 14.
Mortality:
no mortality observed
Description (incidence):
There were no premature deaths throughout the duration of study
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A marked and biologically relevant mean body weight loss of -16g was evident during the first two days of treatment (Days 6 to 8 of gestation) at 80 mg/kg/day (individual body weight losses were -7 g to -32 g). Subsequent mean body weight gain was similar to Control from Day 8. Overall body weight gain (Days 6-20) at 80 mg/kg/day was slightly, but statistically significantly low (82% of Control (p<0.01)).
Body weight gain in females receiving 10 or 30 mg/kg/day was unaffected by treatment.

Gravid uterine weight was unaffected by treatment at 10, 30 or 80 mg/kg/day.
Adjusted body weight change was low at 10 or 30 mg/kg/day (both 65% of Control); adjusted body weight change was markedly low and adverse at 80 mg/kg/day (47% of Control); statistical significance (all p<0.01) was attained by all groups.

PLEASE REFER TO THE ATTACHED TABLES: "BODY WEIGHT AND BODY WEIGHT CHANGES - GROUP MEAN VALUES DURING GESTATION"
"GRAVID UTERINE WEIGHT, BODY WEIGHT AND ADJUSTED BODY WEIGHT CHANGE - GROUP MEAN VALUES ON DAY 20 OF GESTATION"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Overall food consumption was statistically significantly low at 80 mg/kg/day (84% of Control (p<0.01)) during the period of treatment (measured Days 6-20 of gestation); the greatest reduction in food intake was seen during Days 6 to 10 of gestation (57% of Control) that was considered adverse, after which food intake improved.
Food consumption at 30 mg/kg/day was marginally, but statistically significantly low during Days 6 to 10 (p<0.01) and Days 18 to 20 (p<0.05) of gestation (91% and 93% of Control, respectively) the small difference from Control was considered not to be adverse.
Food consumption at 10 mg/kg/day was unaffected by treatment.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Absolute and adjusted thyroid weight was unaffected by treatment at 10, 30 or 80 mg/kg/day.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment related macroscopic findings in adults at 10, 30 or 80 mg/kg/day.
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including Control), and/or were as expected for Sprague Dawley rats of this age; therefore, they were considered not Ethyldiisopropylamine-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related microscopic findings in the thyroid at 10, 30 or 80 mg/kg/day.
All microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including Control), and/or their severity was as expected for Sprague Dawley rats of this age. Consequently, they were considered not Ethyldiisopropylamine-related.
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
Rats do not abort
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The numbers of implantations, resorptions (early/late), pre- and post-implantation losses (%) and live young and the ratio of male to female fetuses were similar to Control and were therefore unaffected by treatment at 10, 30 or 80 mg/kg/day.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
The numbers of implantations, resorptions (early/late), pre- and post-implantation losses (%) and live young and the ratio of male to female fetuses were similar to Control and were therefore unaffected by treatment at 10, 30 or 80 mg/kg/day.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The numbers of implantations, resorptions (early/late), pre- and post-implantation losses (%) and live young and the ratio of male to female fetuses were similar to Control and were therefore unaffected by treatment at 10, 30 or 80 mg/kg/day.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
overall fetal weights were unaffected by treatment at 10, 30 or 80 mg/kg/day.

PLEASE REFER TO THE ATTCHED TABLE: "PLACENTAL, LITTER AND FETAL WEIGH - GROUP MEAN VALUES ON DAY 20 OF GESTATION"
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
All animals surviving to scheduled termination were pregnant. The numbers of implantations, resorptions (early/late), pre- and post-implantation losses (%) and live young and the ratio of male to female fetuses were similar to Control and were therefore unaffected by treatment at 10, 30 or 80 mg/kg/day.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
All animals surviving to scheduled termination were pregnant. The numbers of implantations, resorptions (early/late), pre- and post-implantation losses (%) and live young and the ratio of male to female fetuses were similar to Control and were therefore unaffected by treatment at 10, 30 or 80 mg/kg/day.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
All animals surviving to scheduled termination were pregnant. The numbers of implantations, resorptions (early/late), pre- and post-implantation losses (%) and live young and the ratio of male to female fetuses were similar to Control and were therefore unaffected by treatment at 10, 30 or 80 mg/kg/day.

PLEASE REFER TO THE ATTCHED TABLE: "PLACENTAL, LITTER AND FETAL WEIGH - GROUP MEAN VALUES ON DAY 20 OF GESTATION"
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
When compared with Control, anogenital distance was unaffected by maternal treatment at 10, 30 or 80 mg/kg/day.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants show no relationship to treatment at 10, 30 or 80 mg/kg/day.

PLEASE REFER TO THE ATTACHED TABLE: FETAL EXAMINATIONS - MAJOR ABNORMALITY FINDINGS - GROUP INCIDENCES"
Skeletal malformations:
no effects observed
Description (incidence and severity):
The incidence of major and minor abnormalities and skeletal variants show no relationship to treatment at 10, 30 or 80 mg/kg/day.
Visceral malformations:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed on embryo fetal development
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
80 mg/kg bw/day (actual dose received)
Treatment related:
no

Formulation Analysis


The mean achieved concentrations for Ethyldiisopropylamine in formulations prepared for the first week of treatment were low, ranging from 8.4% (Group 2 (10 mg/kg/day) to 91.6% (Group 4) of nominal. It was considered that the test item evaporated, due to its high volatility into the headspace above the 1 mL sample taken for analysis in a 20 mL vial. 


To prevent potential losses due to headspace in the sample vial, the remaining formulations prepared for the study were sampled directly into volumetric flasks, containing diluent water. The mean analyzed concentrations of the 2nd and 3rd formulations were within -15%/+10% of nominal concentrations, confirming the accuracy of formulation. It was therefore concluded that formulations had been prepared accurately on the study.


The difference from mean remained within 3.3%, confirming precise analysis.


Analyzed Concentrations for Ethyldiisopropylamine in 1% Methylcellulose plus 0.1% Tween 80























































































































































Occasion



Group



Nominal concentration (mg/mL)



Analyzed concentration


(mg/mL)



RME (%)



Difference from mean (%)



Procedural recoveries (%)



Analysis 1



Analysis 2



Mean



At analysis



Mean



14Sep2020



1



0



ND



ND



ND



-



-



-



-



2



5 1



0.429



0.406



0.418



-91.6



±2.75



98.2



99.2



3



15 1



7.32



7.06



7.19



-52.1



±1.81



102.0



4



40 1



33.0



32.2



32.6



-18.5



±1.23



97.3



17Sep2020



1



0



ND



ND



ND



-



-



-



-



2



5 2



4.45



4.41



4.43



-11.4



±0.45



99.0



101.3



3



15 2



15.7



16.7



16.2



+8.0



±3.09



101.3



4



40 2



39.4



39.5



39.5



-.1.3



±0.13



103.5



23Sep2020



1



0



ND



ND



ND



-



-



-



-



2



5 2



4.43



4.51



4.47



-10.6



±0.89



97.8



94.0



3



15 2



13.7



14.3



14.0



-6.7



±2.14



95.3



4



40 2



35.8



38.2



37.0



-7.5



±3.24



88.8



RME      Relative mean error, representing the deviation from nominal


ND          Not detected


1              Samples (1 mL) supplied in 20 mL glass vials.  It is thought that the low analyzed concentrations were due to losses of Ethyldiisopropylamine to the headspace in the vials


2              Samples (1 mL) transferred directly into volumetric flasks containing diluent water, in order to prevent losses to headspace


Mean analyzed concentrations were calculated using unrounded figures.


 Thyroid Hormone Analysis


Attachment 13.3, Attachment 13.4, Attachment 13.6


On Day 20 of gestation, mean serum T3 and T4 and TSH concentrations were statistically significantly low (81%, 81% and 67% of Control, respectively) at 80 mg/kg/day. These were considered non-adverse (See Section 7).


Summary of adult thyroid hormone results on Day 20 of gestation and the HCD ranges





















































Group



1



2



3



4



Dose (mg/kg bw/day)



0



10



30



80



Triiodothyronine (pg/mL)



383



352



374



310**



HCD (pg/mL)



290 - 643



Thyroxine (pg/mL)



12100



11100



11300



9790*



HCD (pg/mL)



8945 - 21790



Thyroid Stimulating Hormone (pg/mL)



1140



928



881



762*



HCD (pg/mL)



374 - 3054



*  p<0.05


**  p<0.01

Conclusions:
The maternal No-Observed-Adverse-Effect-Level (NOAEL) was 30 mg/kg bw/day, based on the adverse loss in maternal body weight during the first two days of treatment (Days 6 and 7 of gestation), the adverse low adjusted body weight change and the low food consumption during Days 6-10 of gestation at 80 mg/kg/day. The embryo-fetal No-Observed-Adverse-Effect-Level (NOAEL) was 80 mg/kg bw/day.

Executive summary:

SUMMARY


The purpose of this study was to assess the influence of Ethyldiisopropylamine (an industrial chemical), on embryo-fetal survival and development in the Sprague Dawley rat, when administered during the organogenesis and fetal growth phases of pregnancy. Three groups of 20 females received Ethyldiisopropylamine at doses of 10, 30 or 80 mg/kg/day by oral gavage administration at a volume dose of 2 mL/kg bwt, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, 1% w/v methylcellulose plus 0.1% Tween 80 at the same volume dose as the treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.


Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating. Gravid uterine and thyroid weights were recorded. A microscopic pathology investigation was performed on the thyroid from all pregnant animals. The ano-genital distance, individual body weight and placental weights were recorded for all fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. Blood samples were taken at necropsy for subsequent thyroid hormone analysis in all pregnant animals.


Results


There were no premature deaths throughout the duration of study.


All animals remained in good clinical condition throughout the study period at 10, 30 or 80 mg/kg/day.


At 30 or 80 mg/kg/day, partially closed eyelids, with or without piloerection were evident from the first day of treatment and persisted in some animals at the end of the day and, at the end of the treatment period (Days 17-19) in some animals before dosing the next day. The incidence of these signs increased during the treatment period. One or two animals at 30 or 80 mg/kg/day were underactive during the first two days of dosing and one animal at 80 mg/kg/day had hunched posture on Day 14 of gestation. There were no signs associated with dosing at 10 mg/kg/day.


A marked and biologically relevant mean body weight loss of -16 g was evident following the first two doses at 80 mg/kg/day (Days 6 and 7 of gestation); overall body weight gain (Days 6-20) was low.


Adjusted body weight change was low at 10 or 30 mg/kg/day and was markedly low and adverse at 80 mg/kg/day; gravid uterine weight was unaffected by treatment at 10, 30 or 80 mg/kg/day.


Food consumption at 80 mg/kg/day was adversely low during Days 6-10 of gestation (57% of Control) and remained slightly low during Days 10-20 of gestation.


Body weight gain in females receiving 10 or 30 mg/kg/day was unaffected by treatment.


Reproductive performance (numbers of resorptions, implantation losses and live young, the ratio of male to female fetuses, placental, total litter or overall fetal weights) and fetal ano-genital distance was unaffected by treatment at 10, 30 or 80 mg/kg/day.


The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment at 10, 30 or 80 mg/kg/day.


On Day 20 of gestation, maternal serum concentrations of Triiodothyronine, Thyroxine and Thyroid Stimulating Hormone were slightly, but statistically significantly low at 80 mg/kg/day, but as absolute and adjusted maternal thyroid weight was unaffected and there were no treatment related microscopic findings in the thyroid at 10, 30 or 80 mg/kg/day and, in the absence of embryo-fetal developmental changes, the findings were considered non-adverse.


Conclusion


The maternal No-Observed-Adverse-Effect-Level (NOAEL) was 30 mg/kg bw/day, based on the adverse loss in maternal body weight during the first two days of treatment (Days 6 and 7 of gestation), the adverse low adjusted body weight change and the low food consumption during Days 6-10 of gestation at 80 mg/kg/day. The embryo-fetal No-Observed-Adverse-Effect-Level (NOAEL) was 80 mg/kg bw/day.


 


 

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
80 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the available data, EDIPA does not warrant classification for reproductive and developmental toxicity according to Regulation (EC) No 1272-2008 and Annex VI of Commission Directive 2001/59/EC

Additional information